1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns the inactivation of ribonucleases (RNases) which can degrade RNA.
2. Description of Related Art
The quality of an RNA preparation greatly affects the results obtained when analyzing it by a number of different molecular biology techniques such as northern blotting, ribonuclease protection assays and RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). Degraded RNA will produce a lower signal than in an equivalent intact RNA sample.
RNA is much more susceptible to degradation than DNA (Sambrook et al., 1989). RNA is readily hydrolyzed when exposed to conditions of high pH, metal cations, high temperatures and contaminating ribonucleases. A major cause of RNA degradation is ribonuclease contamination, and this must be guarded against in virtually all RNA-related procedures, including RNA isolation, mRNA purification, RNA storage, northern blotting, nuclease protection assays, RT-PCR, in vitro transcription and/or translation and RNA diagnostics. In addition to the endogenous ribonucleases from cells and tissues, finger grease and bacteria and/or fungi in airborne dust particles are common sources of ribonuclease. To minimize ribonuclease contamination, appropriate precautions must be followed when handling RNA (Blumberg, 1987; Wu, 1997).
Ribonucleases are difficult to inactivate. For example, while at 90xc2x0 C., bovine pancreatic ribonuclease A (RNase A) has no activity. However, if the enzyme is quickly cooled to 25xc2x0 C., the activity is fully restored. This process is known as reversible thermal denaturation. If the RNase A is incubated at 90xc2x0 C. over time, then decreasing fractions of the activity are recovered at 25xc2x0 C. This process is known as irreversible thermoinactivation. At 90xc2x0 C., it takes several hours to inactivate RNase A (Zale and Klibanov, 1986). Much of the lost activity is attributed to disulfide interchange (Zale and Klibanov, 1986). Further, the inventors and others have found that ribonucleases can even withstand autoclaving (121xc2x0 C., 15 psi, 15 minutes) to some degree. Spackman et al. (1960) tested the stability of RNase A and concluded that it was stable to heat, extremes of pH, and the protein denaturant, urea, results emphasizing the difficulty researchers have had inactivating ribonucleases. For the above reason, a variety of methods other than heating have been developed to inhibit or inactivate ribonucleases. These methods, and their disadvantages, are described below.
In one method of destroying RNases, diethyl pyrocarbonate (DEPC) is added to final concentration of 0.1% to molecular biology reagents, glassware or electrophoresis apparatus, followed by incubating at 37xc2x0 C. for several hours and then autoclaving for 15-20 minutes to destroy the DEPC (Wolf et al., 1970). DEPC reacts with the xcex5-amino groups of lysine and the carboxylic groups of aspartate and glutamate both intra- and intermolecularly (Wolf et al., 1970). This chemical reaction forms polymers of the ribonuclease. However, there are several disadvantages to using DEPC: (1) It is a possible carcinogen and is hazardous to humans; (2) some commonly used molecular biology reagents such as Tris react with and inactivate DEPC; (3) treatment of samples with DEPC is time-consuming; (4) DEPC reacts with the adenine residues of RNA, rendering it inactive in in vitro translation reactions (Blumberg, 1987) and 5) If all of the DEPC is not destroyed by autoclaving, remaining trace amounts may inhibit subsequent enzymatic reactions.
Traditionally, RNA is stored in DEPC-treated water or TE buffer. However, the RNA is not protected from degradation if the sample or the storage solution has a minor ribonuclease contamination. It has been suggested that RNA be stored in ethanol or formamide to protect an RNA sample from degradation because these environments minimize ribonuclease activity (Chomczynski, 1992). The obvious disadvantage is that the RNA sample cannot be directly utilized for analysis or enzymatic reactions unless the ethanol or formamide is removed.
Guanidinium thiocyanate is commonly used to inhibit RNases during RNA isolation (Chomczynski and Sacchi, 1987; Sambrook et al., 1989). A high concentration of guanidinium thiocyanate combined with xcex2-mercaptoethanol is used to isolate RNA from tissues, even those that are rich in ribonucleases, such as pancreas (Chirgwin et al., 1979). Guanidinium is an effective inhibitor of most enzymes due to its chaotropic nature. However, if RNA is dissolved in guanidinium, then it must first be purified from the guanidinium prior to being used in an enzymatic reaction.
Vanadyl-ribonucleoside complexes (VRC) may be used to inhibit RNases during RNA preparation (Berger and Birkenmeier, 1979). The drawback to using VRC, is that VRC strongly inhibits the translation of mRNA in cell-free systems and must be removed from RNA samples by phenol extraction (Sambrook et al., 1989).
Favaloro et al. (1980) employed macaloid, a clay, to absorb RNases. A limitation of this method is that it is difficult to completely remove the clay from RNA samples. Other reagents have been used to inhibit ribonucleases including SDS, EDTA, proteinase K, heparin, hydroxylamine-oxygen-cupric ion, bentonite and ammonium sulfate (Allewell and Sama, 1974; Jocoli and Ronald, 1973; Lin, 1972; Jones, 1976; Mendelsohn and Young, 1978). None of these reagents are strong inhibitors alone, although their inhibitory effect may be improved by using them in combination. Like many of the RNase inhibitors already described, although these chemicals inhibit RNase activity, they also may inhibit other enzymes such as reverse transcriptase and DNase I. Therefore, the RNA must be purified away from the inhibitory reagent(s) before it can be subjected to other enzymatic processes.
Two types of proteinaceous RNase inhibitors are commercially available: human placental ribonuclease inhibitor (Blackburn et al., 1977) and PRIME Inhibitor(trademark) (Murphy et al., 1995). RNases of the class A family bind tightly to these protein inhibitors and form noncovalent complexes that are enzymatically inactive. The major disadvantage of these inhibitors is that they have a narrow spectrum of specificity. They do not inhibit other classes of RNases. Another disadvantage when using placental ribonuclease inhibitor is that it denatures within hours at 37xc2x0 C., releasing the bound ribonuclease. Thus, the RNA sample is only protected for only a few hours at most.
Reducing agents are frequently used as adjuvants to RNA isolation solutions in conjunction with denaturants to reduce the disulfide bonds in RNases that are rendered accessible by the denaturant. Commonly used reducing reagents are xcex2-mercaptoethanol, dithiothreitol (DTT), dithioerythritol (DTE), and glutathione. Another such reducing agent is the amino acid cysteine. xcex2-mercaptoethanol is often included in RNA isolation solutions combined with guanidinium thiocyanate to reduce ribonuclease activity and solubolize proteins (Chomcyznski and Sacchi, 1987). DTT is the strongest reducing reagent of the three listed.
DTT has low redox potential (xe2x88x920.33 volts at pH 7.0) and is capable of maintaining monothiols effectively in the reduced form and of reducing disulfides quantitatively (Cleland, 1964). DTT acts as a protective agent for free sulfhydryl groups. It is highly water soluble, with little tendency to be oxidized directly by air, and is superior to other thiols used as protective reagents. DTT""s reducing activity can be accurately assayed using 5, 5xe2x80x2-dithiobis (2-nitrobenzoic acid) or DTNB (Cleland, 1964). The reduction of DTNB mediated by DTT generates a yellow color whose absorbance can be measured at 412 nm using a spectrophotometer. RNase A, RNase 1 and RNase T1 all contain disulfide bonds (Ryle and Anfinesen, 1957; Barnard, 1969) and, therefore, are susceptible to reduction.
DTT has been used as an inhibitor of RNase A in the isolation of polyribosomes (Boshes, 1970; Aliaga, 1975). In Boshes"" experiment, polyribosome preparations were treated with RNase A (10 xcexcg/ml) in solution A (10 mM MgCl2: 10 mM Tris [pH 7.6]: 50 mM KCl) in the presence or absence of 4 mM DTT at 4xc2x0 C. for 20 minutes. The treatment of polyribosomes with RNase A generated monoribosomes. Boshes observed that polyribosomes treated with RNase A in the presence of 4 mM DTT reduced the conversion of polyribosomes to monoribosomes and from that result he concluded that DTT was an RNase inhibitor. Boshes"" statement was based on the effect of DTT on the conversion of polyribosomes to monoribosomes by RNase A. He did not directly assay the degradation of purified RNA. Since Boshes was working with a complex, uncharacterized protein mixture, it is unclear as to what may have been responsible for the decreased production of monoribosomes. For example, the addition of DTT may have increased the activity of the endogenous mammalian RNase inhibitors rather than act directly on the RNases. These RNase inhibitors require a reducing environment for activity.
Heat has been used to inactivate RNase A by mediating the breakage of disulfide bonds. Zale and Klibanov (1986) performed inactivation of RNase A at 90xc2x0 C. and pH 6.0 for 1 hour, which induced the following chemical changes: disulfide interchange, xcex2-elimination of cysteine residues, and deamidation of asparagine. This type of heat treatment did not completely inactivate the ribonuclease. A major disadvantage is that a long-term, high-temperature treatment (90-100xc2x0 C.) is incompatible with RNA. Such treatment promotes the hydrolysis of RNA. In fact, the inventors have found that total RNA incubated at 65xc2x0 C. for several hours is almost completely degraded. Thus, treating an RNase sample with extreme heat to inactivate ribonucleases will mediate the destruction of the RNA that the user is trying to protect.
The present invention provides a general method for rapidly inactivating ribonucleases and RNA storage solutions adapted for use in such methods. These methods comprise the steps of obtaining a composition; obtaining a reducing agent; admixing the reducing reagent and sample; and heating. A composition is understood to be any composition that may contain RNA, including, but not limited to, a solution, a sample, a reagent, and an extract, such as a crude cellular extract.
The inventors"" method for inactivating ribonuclease can protect RNA from ribonuclease degradation during storage. More importantly, an RNA-containing compositions can be used immediately after ribonuclease inactivation for RNA analysis, and in enzymatic reactions such as the synthesis of cDNA by reverse transcriptase and the degradation cellular DNA by a deoxyribonuclease such as DNase I.
As used herein, the terms xe2x80x9cRNase inactivationxe2x80x9d or the xe2x80x9cinactivation of RNasesxe2x80x9d denotes that there is no detectable degradation of the sample RNA under the assay conditions used. Similarly, the term xe2x80x9csubstantially inactivatedxe2x80x9d connotes that there is no substantial degradation of RNA detected in a composition that may contain RNA. xe2x80x9cSubstantialxe2x80x9d degredation is defined as degradation that would impare the use of the RNA in the types of protocols described in this specification. As used herein, the term xe2x80x9cinhibitionxe2x80x9d of ribonuclease activity means that activity of at least one ribonuclease is reduced in a sample treated according to the invention relative to a sample not treated according t the invention. xe2x80x9cInhibitionxe2x80x9d does not require RNase inactivation or even subatantial RNase inactivation.
In broad aspects, the present invention relates to methods for inhibiting the activity of ribonucleases that may be present in a composition comprising: (a) obtaining a composition; (b) obtaining a reducing agent; (c) admixing the composition and the reducing agent; and (d) heating the admixture; such that any activity of any ribonucleases in the composition is inhibited. The reducing agent, concentrations of components and heating conditions may be, for example, the conditions discussed elsewhere in this specification.
In one embodiment, the present invention relates to reagents for use in methods for inactivating or inhibiting ribonucleases and such methods comprising: (a) obtaining a composition that may contain a ribonuclease; (b) obtaining a reducing agent; (c) admixing the composition and the reducing agent; and (d) heating the admixture, wherein ribonucleases in the composition are inactivated or inhibited. There are many different manners in which the methods and reagents of the present invention may be used. However, in a preferred embodiment, the reagents and methods will be used to inactivate or inhibit any ribonucleases that are present in a composition that is a reagent used in molecular biology, either in cases where ribonuclease contamination is known or expected to have occurred, or simply as an additional prophylactic step in attempts to avoid RNase contamination. As discussed above, ribonuclease contamination can seriously affect the results of molecular biological assays. Therefore, there is great value in having simple, efficient methods for preventing ribonuclease contamination of molecular biology reagents. In many cases, the molecular biology reagent will be one employed in the handling of RNA, water, TE buffer, 20xc3x97SSC, 10xc3x97MOPS, Tris buffer, EDTA, nucleic acid hybridization buffer, sodium acetate buffer, formalin tissue fixative, in situ hybridization buffer, or nucleic acid storage buffer/solution.
Many agents and reagents having reducing properties are well known to those of skill in the art, and, by employing assays described herein, one of ordinary skill in the art will be able to determine which of any of these reducing agents will be of use in the present invention. Presently preferred reducing agents are those comprising DTT, xcex2-mercaptoethanol, cysteine, or dithioerithritol. The final concentration of these reducing agents in the composition that is to be protected from ribonuclease can be any that functions to achieve the ribonuclease protective activity that is the purpose of the invention. Presently preferred concentrations are those between 0.5 and 500 mM reducing agent in the admixture of the sample and the reducing agent. More presently preferred concentrations are between 1 and 200 mM in the admixture. Even more presently preferred concentrations are between 2 and 60 mM. The presently most preferred concentration is 20 mM in the admixture. Of course, the invention is in no way limited to these preferred concentrations.
In most cases, the reducing agent is comprised in a buffer solution prior to admixing. The buffer solution will comprise the agent at a high enough concentration such that the final concentration of the agent in the admixture is sufficient to realize the ribonuclease protective goals of the invention. The buffer solution may also comprise a chelator such as sodium citrate, EGTA, or EDTA.
The admixture may be heated to any temperature and for any amount of time that is sufficient to accomplish the ribonuclease inactivation and/or inhibition goals of the invention. Presently preferred conditions call of the heating of the admixture to at least 37xc2x0 C. for at least 3 minutes. Heating may be accomplished by any means standardly employed in a biological lab. The inventors typically use a heat block or water bath that has been set to the desired temperature.
One of the advantages of the present invention is that the methods do not affect the stability of any RNA that is comprised in the composition that is being treated to inactivate and/or inhibit ribonucleases. Therefore, a specific embodiment of the invention comprises methods for inactivating/inhibiting ribonucleases in the presence of RNA comprising: (a) obtaining a composition comprising RNA; (b) obtaining a reducing agent; (c) admixing the composition and the reducing agent; and (d) heating the admixture, wherein ribonucleases in the composition are inactivated and/or inibited. The RNA composition may be any composition that contains RNA, including but not limited to a tissue sample, a cell sample, a crude cell preparation, isolated total RNA, and any form of purified RNA. In a preferred embodiment, the composition is comprised of purified RNA. The reducing agents, other components, concentrations, times, and temperatures are typically as described above.
In some embodiments, the invention relates to methods for sequential inactivation or inhibition of any ribonucleases in a composition such that any ribonucleases introduced to the sample at some time after a first inactivation or inhibition procedure may be inactivated or inhibited comprising: a) obtaining a composition; b) obtaining a reducing agent; c) admixing the composition and the reducing agent; d) performing a first heating of the admixture, whereby any ribonucleases in the admixture are inactivated or inhibited; e) determining that a further inactivation or inhibition procedure is warranted to inactivate any ribonucleases that may have been introduced to the sample subsequent to the first heating; f) performing a second heating of the admixture, whereby ribonucleases in the admixture are inactivated. The benefits of these methods are clear. One may have a composition comprising RNA that also contains an appropriate amount of reducing agent. Prior to storage of the RNA, one can simply heat the composition for an appropriate length of time to an appropriate temperature, thereby inactivating or inhibiting any ribonucleases. The next time the RNA is accessed, the remaining RNA may be reheated to inactivate or inhibit any ribonucleases that might have been introduced into the composition by the access procedure. This process may be carried out an indefinite number of times throughout the storage life of the RNA.
The present invention also comprises solutions for storing RNA comprising: a) a reducing agent; and b) an RNA sample. In some preferred embodiments, the reducing agent will be selected from the group consisting of DTT, dithioerythritol, xcex2-mercaptoethanol, and cysteine. The final concentration of the reducing agent in the solution can be any concentration that functions to achieve the ribonuclease protective activity that is the purpose of the invention upon heating. Presently preferred concentration are those between 0.5 and 500 mM reducing agent in the solution. More presently preferred concentrations are between 1 and 200 mM in the solution. Even more presently preferred concentrations are between 2 and 60 mM. The presently most preferred concentration is 20 mM in the solution. Of course, the invention is in no way limited to these preferred concentrations. Some preferred RNA storage solution comprise a buffer, and presently preferred pHs of the solution are between 5.0-7.0. High pHs mediate the hydrolysis of RNA. However, any pH that does not adversely affect the RNA may be used. The solution may also comprise a metal chelator such as, for example, sodium citrate, EGTA, or EDTA. The chelator is sometimes desired because some metal cations can mediate the hydrolysis of RNA in a sample.
In some general aspects, the invention relates to method for inactivating or inhibiting ribonucleases in a cell sample comprising: obtaining a cell sample; obtaining a reducing agent; admixing the cell sample and the reducing agent, and heating the admixture; wherein any ribonucleases in the cell sample are substantially inactivated and/or inhibited.
In addition, the present invention encompasses methods for producing cDNA from a crude cellular extract, which takes advantage of the ribonuclease inactivation and/or inhibition methods described above and herein. A crude cellular extract can be derived from any cell type, including cell lines and cells derived from organs, and can be prepared according to standard methods known to those of skill in the art. In some examples, cDNA can be produced from a crude cellular extract without isolating RNA from the extract. A specific example of these methods comprises: (a) obtaining a crude cellular extract; (b) obtaining a reducing agent; (c) admixing the crude cellular extract and the reducing agent; (d) heating the admixture, wherein any ribonucleases in the crude cellular extract are substantially inactivated or inhibited; (e) incubating the admixture with reverse transcriptase under conditions to allow reverse transcription; and, (f) amplifying the products of the reverse transcription. In another example of these methods, the admixture is also incubated with a deoxyribonuclease, such as DNase I, prior to the reverse transcription reaction. In preferred embodiments at this time, the reducing agent is DTT, xcex2-mercaptoethanol, cysteine, or dithioerithritol. In even more preferred embodiments, the reducing agent is DTT, however, the invention should not be limited to the preferred embodiments. When DTT is employed in a method for producing cDNA from a crude cellular extract in some examples, the final concentration of the DTT is between 1 and 200 mM in the admixture. Furthermore, in preferred examples, the admixture is heated to at least 37xc2x0 C., and in some embodiments, the admixture is heated for at least 3 minutes.
The invention is further understood to encompass kits, in a suitable container, for producing cDNA from a cell sample. The kit can contain components that can be used for any portion of the method for preparing cDNA from a cell sample. A kit of the present invention could comprise a reducing agent and a buffer. Such a kit could also include components used for reverse transcription and/or components used to amplify nucleic acid sequences. It is further contemplated that a kit of the present invention includes reagents that may be useful, but not necessary for either reverse transcription and/or nucleic acid amplification. These reagents include water, such as RNase-free dH2O.
In one embodiment, the kit contains at least one of the following: a buffer, such as PBS; a reducing agent; a reverse transcription buffer; reverse transcriptase; dNTP mix (comprising at least one of the dNTPS of ATP, GTP, CTP, and TTP); a deoxyribonuclease such as DNase I, which may be RNase-free; an RNase inhibitor; and Lambda ARMORED RNA(copyright). In another example, the kit for preparing cDNA from a cell sample comprises a buffer, such as PBS; and/or a reducing agent; and/or a reverse transcription buffer; and/or a reverse transcriptase; and/or a dNTP mix (comprising at least one of the dNTPS of ATP, GTP, CTP, and TTP); and/or a deoxyribonuclease such as DNase I, which also may be RNase-free; and/or an RNase inhibitor, such Placental RNase Inhibitor; and/or Lambda ARMORED RNA(copyright). It is also contemplated the kits of the present invention may include primers such as deoxynucleotide oligomers, and reagents for implementing positive and negative controls. An example of a primer that can be used is Oligo dT18 primer or Random Decamer Primer. Specific primers to amplify specific nucleotide sequences may also be included in a kit of the present invention.
Following long-standing patent law, the words xe2x80x9caxe2x80x9d and xe2x80x9can,xe2x80x9d when used in conjunction with the word xe2x80x9ccomprisingxe2x80x9d in the claims or specification, denote one or more.